The techniques of genetic engineering allow for the introduction of specific genetic information into a host regardless of the source of such information. Accordingly, it is possible to add foreign derived information to a host's genetic material. Moreover, it is possible to add additional copies of endogenous genetic material to a host. In either case, if the additionally provided genetic information introduced into the host is expressed, the result of such expression is to confer and/or enhance a desired trait or traits.
Expression of added genetic information in a host may be regulated by either endogenous regulatory elements or regulatory elements incorporated into the host with the added material. In order for expression of added genetic information to be controlled by endogenous regulatory elements, the added material must recombine with the host's genetic material in the correct orientation and at a location in the host's genetic material which contains such regulatory elements. Alternatively, the regulatory elements may be introduced into the host with the genetic material to be expressed.
Several genes may be added to a host, either simultaneously or at different times. If done simultaneously, the added material may be separate or linked. In all cases, in order for added genetic material to be expressed, it must be linked to regulatory genetic sequences which mediate gene expression.
It is more effecient, especially when adding more than one gene to a host, to link regulatory sequences to each gene rather than rely on use of endogenous regulatory sequences. In cases where the host is to provide the regulatory elements, the probability of correct integration of all added genes in each host cell is greatly reduced when more than one gene is sought to be incorporated since the requirements for functional integration operate independantly for each gene. If separate genes, each containing the necessary regulatory sequences are added to a host cell population, the probability of a cell being transformed by functioning copies of each desired genes is higher because the number of locations in the host cell's genetic material in which the added material can integrate is far greater.
When genetic information is added to a host, the cells which have incorporated the genetic material and are capable of expressing it must be selected. Selection of transformed host cells from the starting material host cell population can be a difficult task. If expression of the desired protein is not easily detected, a marker gene is usually added together with the gene sought to be expressed. The marker gene, with its own regulatory sequences, is usually linked to the desired gene to confirm the presence of the desired gene in selected cells. The difficulties associated with selecting transformed host cells are compounded when if addition of more than one gene is performed and the desired genes are not linked to each other because different markers must be used to detect the presence of each new gene. When two genes are sought to be incorporated, this can be very inconvenient. When more than two genes are to be selected, this strategy becomes increasingly unfeasible.
One method used to overcome the difficulties of multiple selections is to link all the genes to each other. Using this approach, each gene is linked sequentially and a marker gene is additionally linked so that a single selection can be made to determine the presence of every gene. In order to proceed following this strategy, it is necessary to provide each linked gene with its own set of regulatory sequences. If each sequentially linked gene is to be equipped with its own set of regulatory sequences and each multiple gene construct additionally contains a marker gene with its own set of regulatory sequences, the size of the construct presents difficulties in both propagating the construction and transforming host cells with it.
The present invention overcomes the problems which are associated with incorporating more then one gene in an expressable form into a host cell by providing a multigene which is comprised of the several desired genes under the regulatory control of a single set of genetic regulatory elements. Thus, the multigene of the present invention is transcribed as a single polyprotein which must be processed subsequently in order to yield the substituent polypeptides.
Expression of genetic material to produce functional proteinaceous molecules generally requires several steps including post-translational modification of the gene product. In cases where the gene expressed is a multigene and therefore the gene product is a polyprotein, the translation product may be cleaved to form a plurality of polypeptides, each representing a desired product which may be functional with or without further modification.
Examples of translational products which are processed into a plurality of polypeptides are the polyproteins encoded by the genetic material of some viruses. These viruses contain a multigene which is expressed as a single gene under the control of a single set of regulatory elements. A single mRNA, generated from transcription of the multigene, is translated into a single polyprotein. The individual genes of the multigene are linked in series with intermediate linking sequences that encode an amino acid sequence which is a proteolytic cleavage site. Thus, the translated polyprotein contains a series of polpeptides which are connected to each other by proteolytic cleavage sites. The polyprotein is processed by an appropriate protease which cleaves the polyprotein at the cleavage sites and thereby severs the polyprotein into individual separate polypeptides. The protease which cleaves the polyprotein at the specific sites may be an endogenous protease or it may be a protease encoded by the viral genetic information.
The present invention provides a multigene which comprises a gene for a protease that recognizes the amino acid sequences encoded by the nucleotide sequences that link the individual genes of a multigene. Accordingly, the single translation product of the multigene is cleaved into the desired polypeptides. Using the present invention, it is possible to introduce a plurality of genes into a host without experiencing the problems associated with alternative methods. The genetic information which is incorporated into hosts using the present invention can be expressed and a plurality of polypeptides encoded therein may be produced.